This application requests a BioRad Multi-photon microscope optimized for quantitative imaging with living cells and tissues. The BioRad Multi- photon laser scanner will be mounted on an Olympus fixed stage upright microscope with water immersion objectives for working either with or without coverslips in aqueous environments, and multi-photon excitation will be via a Spectra-Physics mode-locked Ti-Sa laser. The facility will serve investigators from multiple departments throughout the Indiana University School of Medicine, who need the instrument to perform live cell and live tissue fluorescence microscopy that is beyond the capabilities of confocal microscopy. Our 4 major users require multi-photon microscopy to (1) provide high resolution imaging of cellular and subcellular events within tissue at focal depths greater than 50mum from the surface (at these depths, confocal microscopy can neither provide high spatial resolution nor avoid photodamage), and (2) permit localized deposition of energy for uncaging or photobleaching experiments so that events can then be tracked by conventional confocal microscopy on the same microscope. All major users have documented that multi-photon microscopes based on the BioRad scanner will provide a significant advance beyond confocal microscopy in addressing their experimental needs. The needs of our users can not be met by equipment at our institution, because there is no multi-photon microscope at the entire Indiana University, and confocal microscopy is inadequate to address the questions of the users. The multi-photon microscope would be installed in a location near the laboratory of the PI which is central to all investigators and would facilitate daily monitoring of equipment status by the PI. The application is supported by substantial interest, need, and financial support at the University, and will serve a broad NIH-funded scientific community.